A major impediment in the therapeutic success of breast cancer (BC) arises from the persistence of clinically undetectable breast cancer stem cells (BCSCs) that need addressal by targeting translationally relevant markers to restrain relapse. Aurora kinase A (AURKA), due to its negative prognostic effect, was considered in clinical trials yet showed an unappreciable response, highlighting the need for additional markers. The present study tried exploring AURKA as a tool of relevance to detect the abundance of Oct4/Sox2(octamer-binding transcription factor 4/sex-determining region Y-box 2)-expressing BCSCs. The purpose was to understand the underlying intricacies governing the limited success of AURKA inhibition and to seek an improved relevant marker profile for detecting disseminated BCSCs. Flow cytometry and chromatin immunoprecipitation assay findings correlated Oct4/Sox2/AURKA expression, proposing an Oct4/Sox2 threshold-dependent AURKA induction. Surprisingly, in BC patient blood, Oct4/Sox2+ve cells were apparently lacking AURKA, emphasizing marker profile dynamicity in disseminating BCSCs with transient cell fate alterations. Immunoprecipitation/immunofluorescence results highlighted an interaction of pAURKA with fate-determinant pNUMB, hinting toward the existence of an AURKA/pNUMB axis for mesenchymal fate induction in breast cancer cells. Oct4/Sox2/AURKA+ve cells further expressed vimentin, highlighting a mesenchymal fate in BCSCs as evident from correlated Oct4/Sox2 and vimentin expression in patient blood. Hence, our study indicated an existing Oct4/Sox2/AURKA/pNUMB axis during transient mesenchymal differentiation of BCSCs and subsequently advocated for a peripheral blood-based approach using AURKA and vimentin for tracking BCSCs, thus supporting a co-targeting strategy. Preliminary in vitro intervention using combinatorial targeting of AURKA and vimentin reduced stemness propensities. Based on these interesting observations, further in-depth studies are warranted for clinical validation.

BACKGROUND:Kinases are emerging as promising targetable choices for Triple negative breast cancer (TNBC) patients who suffer from lack of targeted therapy. Aurora kinase A (AURKA) inhibitor alisertib although showed promise but failed to impart any ultimate benefit in TNBCs. Considering initial preclinical success of alisertib, this study tried to explore the underlying mechanisms of its subsequent insensitivity in TNBCs. METHODS:Breast cancer kinome was screened for kinases with high clinical significance by clinical kinase indexing coupled with studying their differential expression pattern and prognostic impact using available datasets. Relevant kinase inhibitors were assessed for pharmacological/toxicological parameters in silico. Alisertib induced polyploid giant cancer cells (PGCCs) were observed microscopically and flow cytometrically. PGCC viability was assessed by single cell MTT and (BrdU) assay. Epithelial and mesenchymal transition (EMT) and stemness markers were examined by immunofluorescence. PGCC enrichment was additionally checked in stained tissue explant/histocultures. Alisertib insensitive cells were developed by repeated alisertib pulsing as confirmed by MTT assay. Alisertib insensitive mammospheres were targeted with mifepristone. RESULTS:Clinical Kinase Index (CKI) scoring and pan cancer expressional profiling coupled with analysis of prognostic effect revealed AURKA as a targetable prognostically relevant kinase. AURKA-targeting agent alisertib demonstrated acceptable pharmacological/toxicological properties but enriched PGCCs in in vitro and tumour histocultures. PGCCs maintained replicative potency producing viable progenies. They retained expression of EMT and stemness markers; demonstrating single cell clonogenicity. PGCC enrichment led to reduced alisertib sensitivity as evident from enrichment of insensitive mammospheres, targetable by mifepristone. CONCLUSION:PGCCs contributed to alisertib insensitivity in TNBCs that may be targeted by mifepristone.

Background: Steroid hormone receptors (HRs) such as estrogen receptor/progesterone receptor are routinely used for stratifying breast cancer (BC) patients during endocrine-targeted therapy, except triple-negative BCs which lack these receptors. Thus, probing for alternative steroid HRs with prognostic value in BC is necessary. Glucocorticoid receptor (GR), another steroid HR, associated with prosurvival has been known to be prognostic in breast carcinoma, although studies in the Indian patients’ cohort are scanty. Antiapoptotic protein B-cell lymphoma 2, (Bcl-2) being positively associated with GR, is also known to have a significant influence on BC progression, but its exact role is paradoxical. Aim and Objective: This study aimed to look into the expressional status of GR in the Indian BC patients’ cohort to understand the molecular basis of its association with Bcl-2 to provide a basis for the prognostic value of Bcl-2 and its impact on clinicopathological parameters related to disease progression. This study was conducted in the BC patient cohort ( n = 15) of all molecular subtypes visiting hospital outpatient department. Materials and Methods: In silico data mining, flow cytometry, enzyme-linked immunosorbent assay, immunoblotting, and co-immunoprecipitation were performed to analyze for subsequent observations. Pearson correlation analysis and Chi-squared tests were performed to derive the statistically relevant findings. Results: GR is co-expressed with Bcl-2 in the concerned cohort, and participates in protein-protein interactions subsequently sustaining its expression and supplementing to aggressiveness of the disease. Conclusion: GR/Bcl-2 interaction in preliminary findings demanded further extensive research to establish GR and Bcl-2 as promising targets in BC.

A BSTRACT Context: Enhancing radiotherapeutic efficacy in tumor cells and sparing the normal tissues are major clinical concerns for the betterment of cancer therapy. Genistein (GEN) being a radiosensitizer ameliorates the effectiveness of radiation-induced cell killing by inducing DNA damage. This molecule is accountable for minimizing radiation-related toxicity and protecting healthy cells. However, the explicit mechanism of action of such molecules needs exploration. Aims: The objective of this study is to investigate the mechanistic action of GEN in cervical cancer cell radiosensitization. Settings and Design: Cervical squamous carcinoma cell SiHa and a radioresistant subline SiHa/RR (developed and isolated from SiHa) were taken for this study. The experiments were performed by pretreating the cells with IC 30 dose of GEN, followed by acute irradiation to detect the impact of GEN in imparting radiosensitivity. Subjects and Methods: Optimal dose selection of GEN was performed by MTT assay, and radiosensitizing potency was determined by pretreating the cells with IC 30 dose of GEN, followed by challenging with acute incremental doses of radiation. Mechanistic parameters were checked by clonogenic assay, cell cycle analysis, DNA damage estimation, apoptosis, and wound healing-sphere-forming assay. Statistical Analysis Used: Statistical analysis was performed in GraphPad software by performing the Student’s t -test. Results: Results depicted decreased numbers of colonies, increased frequency of DNA damage and apoptotic cells, and suppressed wound healing ability along with restrained sphere-forming ability upon the intervention of cells with GEN before radiation exposure. Such observations implied that GEN pretreatment renders improved radiosensitivity in cervical cancer by increased DNA damage-mediated G2/M arrest with subsequent apoptosis. Conclusions: GEN by inducing DNA damage stimulates radiation-induced cell killing in vitro .

Recent therapeutic advances have significantly uplifted the quality of life in breast cancer patients, yet several impediments block the road to disease-free survival. This involves unresponsiveness towards administered therapy, epithelial to mesenchymal transition, and metastatic progression with the eventual appearance of recurrent disease. Attainment of such characteristics is a huge adaptive challenge to which tumour cells respond by acquiring diverse phenotypically plastic states. Several signalling networks and mediators are involved in such a process. Glucocorticoid receptor being a mediator of stress response imparts prognostic significance in the context of breast carcinoma. Involvement of the glucocorticoid receptor in the signalling cascade of breast cancer phenotypic plasticity needs further elucidation. This review attempted to shed light on the inter-regulatory interactions of the glucocorticoid receptor with the mediators of the plasticity program in breast cancer; which may provide a hint for strategizing therapeutics against the glucocorticoid/glucocorticoid receptor axis so as to modulate phenotypic plasticity in breast carcinoma.

Aurora Kinase A being overexpressed in majority of cancers, appear to be an attractive therapeutic target. However, a Phase III clinical trial of Alisertib, a selective AURKA inhibitor, resulted in no better response compared to the comparator arm of chemotherapeutic regimen raising question regarding ability of the same to target the undetectable stem cell functions. In silico analysis indicated regulation of AURKA by the stemness factors Oct4 and Sox2. TCGA data indicated positive correlation of each of the factors with AURKA which were eventually validated in cell lines, patient tissues and blood by flow cytometry along with Oct4 binding on AURKA promoter being detected by ChIP assay. However, indirect immunofluorescence and cell cycle analyses indicated proliferation-independent AURKA functions during asymmetric cell division, a characteristic feature of stem cells. Thorough screening of the AURKA positive cells in patient samples denoted epithelial to mesenchymal transition and significant upregulation of Vimentin, a mesenchymal marker and ABCG2, a drug resistance marker under Oct4 or Sox2 influence. Overall, our study demonstrated combinatorial selection Oct4, Sox2, AURKA, Vimentin and ABCG2 for diagnostics and intervention of circulating breast cancer stem cells as a blood-based, cost-effective and simple approach which will be beneficial in reducing relapse.

Neutrophils are the most abundant immune cells and make up about 70% of white blood cells in human blood and play a critical role as the first line of defense in the innate immune response. They also help regulate the inflammatory environment to promote tissue repair. However, in cancer, neutrophils can be manipulated by tumors to either promote or hinder tumor growth depending on the cytokine pool. Studies have shown that tumor-bearing mice have increased levels of neutrophils in peripheral circulation and that neutrophil-derived exosomes can deliver various cargos, including lncRNA and miRNA, which contribute to tumor growth and degradation of extracellular matrix. Exosomes derived from immune cells generally possess anti-tumor activities and induce tumor-cell apoptosis by delivering cytotoxic proteins, ROS generation, H2O2 or activation of Fas-mediated apoptosis in target cells. Engineered exosome-like nanovesicles have been developed to deliver chemotherapeutic drugs precisely to tumor cells. However, tumor-derived exosomes can aggravate cancer-associated thrombosis through the formation of neutrophil extracellular traps. Despite the advancements in neutrophil-related research, a detailed understanding of tumor-neutrophil crosstalk is still lacking and remains a major barrier in developing neutrophil-based or targeted therapy. This review will focus on the communication pathways between tumors and neutrophils, and the role of neutrophil-derived exosomes (NDEs) in tumor growth. Additionally, potential strategies to manipulate NDEs for therapeutic purposes will be discussed.

BACKGROUND/AIM:Compromised cell-cycle checkpoint is a major obstacle for rendering radiotherapeutic success of radioresistant cells. Aspirin (ASA), an anti-inflammatory agent was repurposed previously for improving radiotherapy by limiting radiation toxicity. However, the underlying mechanism was unclear. The present study aimed to identify the mechanism of ASA mediated reversal of radioresistance in cervical cancer cells.METHODS:Radioresistant subline SiHa/RR was developed from parental cervical squamous carcinoma cell line SiHa by chronic fractionated irradiation (IR). The radioresistance property of SiHa/RR was confirmed by clonogenic assay. Alteration in cell-cycle by ASA was determined by flow cytometry. ASA induced nuclear damage as consequence of mitotic catastrophe was confirmed by microscopic observation. The interaction between ASA and G2/M regulators was explored through in silico docking analysis and expressional change of them was affirmed by western blotting. Immunofluorescence study to examine Aurora Kinase A localization in presence and absence of ASA treatment was conducted. Finally the radiosensitizing ability of ASA was verified by apoptotic parameters (flow cytometrically and by western blotting).RESULT:Higher colony forming ability of SiHa/RR compared to SiHa became restrained upon ASA (5μM) treatment prior to IR. Flow cytometric analysis of ASA treated cells showed increased G2/M population followed by enlargement of cells displaying giant multinucleated morphology; typical characteristics of mitotic catastrophe. Underlying noteworthy mechanisms involved decreased expressions of G2/M regulatory proteins (Cyclin B1, CDK1, Aurora A Kinase, pAurora A Kinase) in IR/ASA along with inhibiting nuclear localization of Aurora Kinase A in SiHa/RR. Docking results also supported the findings. Prolonged treatment (12 h) with ASA led to apoptosis by altering expressions of Bcl2, Bax and Cytochrome C; which was achieved through the event of mitotic catastrophe.CONCLUSION:This work established that G2/M arrest and mitotic catastrophe can be considered as the principle mechanism of restoration of radiosensitivity in SiHa/RR by ASA pretreatment.

Acquired cisplatin resistance in cervical cancer therapy is principally caused by reduction in intracellular drug accumulation, which is exerted by hyperactivation of the oncogenic PI3K/Akt signaling axis and overexpression of cisplatin-exporter MRP2 along with prosurvival effectors NF-κB and IAPs in cervical cancer cells. These activated prosurvival signaling cascades drive drug efflux and evasion of apoptosis for rendering drug-resistant phenotypes. Our study challenges the PI3K/Akt axis in a cisplatin-resistant cervical cancer scenario with phenethylisothiocyanate (PEITC) for chemosensitization of SiHaR, a cisplatin-resistant sub-line of SiHa and 3-methylcholanthrene–induced cervical cancer mice models. SiHaR exhibited higher MRP2, p-AktThr308, NF-κB, XIAP, and survivin expressions which cumulatively compromised cisplatin retention capacity and accumulated PEITC better than SiHa. SiHaR appeared to favor PEITC uptake as its accumulation rates were found to be positively correlated with MRP2 expressions. PEITC treatment in SiHaR for 3 h prior to cisplatin exposure revived intracellular platinum levels, reduced free GSH levels, generated greater ROS, and altered mitochondrial membrane potential compared to SiHa. Western blot and immunofluorescence results indicated that PEITC successfully downregulated MRP2 in addition to suppressing p-AktThr308, XIAP, survivin, and NF-κB expressions. In mice models, administration of 5 mg/kg body-weight PEITC priming dosage prior to treatment with 3 mg/kg body-weight of cisplatin remediated cervical histology and induced tumor regression in contrast to the group receiving the same dosage of cisplatin only. This suggested PEITC as a potential chemosensitizing agent in light of acquired cisplatin resistance in cervical cancer and established its candidature for Phase I clinical trial.

Cervical cancer being one of the leading gynecological cancers, poses a major threat by its ever-increasing trend of global recurrence. Radioresistance, one of the major challenges confronted during the treatment of cervical cancer is manifested by increased rate of cellular proliferation, migration, invasion and alterations in cell cycle. Aurora Kinase A (AURKA), a mitotic serine/threonine kinase was found to be overexpressed in cancers and is associated with development of acquired therapy resistance. The principal objective of this study was to explore the mechanisms by which AURKA confers radioadaptive response in cervical cancer cells. Parental cervical squamous carcinoma cell line SiHa was subjected to recurrent challenge towards fractionated dose of X-irradiation. Finally, a resistant subline (SiHa/RR) was isolated at 40Gy. SiHa/RR exhibited higher expression of AURKA/ pAURKA along with activation of the signaling pathways (HIF1α, pAkt, NFκB) vis-à-vis lower expressions of the proteins (p53, Gadd45a), which are generally suppressed by AURKA. Interestingly, inhibition of AURKA in SiHa/RR showed improved radiosensitivity by reducing cell viability, restrained wound healing capacity as well as sphere forming ability and accelerated radiation induced apoptosis. Ectopic overexpression of AURKA gave rise to radioresistant phenotype in parental SiHa by stimulating nuclear translocation of NFκB. This pattern of increased nuclear localization of NFκB was also observed in resistant subline as a consequence of activation and overexpression of AURKA. These findings strengthened the active involvement of AURKA in radioresistance via driving NFκB mediated signaling pathway to deliver radioresistant associated adaptive complexities.

The clinical scenario of acquired cisplatin resistance is considered as a major impediment in cervical cancer treatment. Bulky drug-DNA adducts formed by cisplatin elicits DNA damage response (DDR) which either subsequently induces apoptosis in the cervical cancer cells or enables them to adapt with drug assault by invigorating pro-survival molecular cascades. When HPV infected cervical cancer cells encounter cisplatin, a complex molecular interaction between deregulated tumor suppressors, DNA damage-repair enzymes, and prosurvival molecules get initiated. Ambiguous molecular triggers allow cancer cells to cull apoptosis by opting for a survival fate. Overriding of the apoptotic cues by the pro-survival cues renders a cisplatin resistant phenotype in the tumor microenvironment. The present review undrapes the impact of deregulated signaling nexus formed due to crosstalk of the key molecules related to cell survival and apoptosis in orchestrating platinum resistance in cervical cancer.

The high abundance of drug efflux pumps in cancer stem cells (CSCs) contributes to chemotherapy resistance. The transcriptional regulator SMAR1 suppresses CSC expansion in colorectal cancer, and increased abundance of SMAR1 is associated with better prognosis. Here, we found in breast tumors that the expression of SMAR1 was decreased in CSCs through the cooperative interaction of the pluripotency factors Oct4 and Sox2 with the histone deacetylase HDAC1. Overexpressing SMAR1 sensitized CSCs to chemotherapy through SMAR1-dependent recruitment of HDAC2 to the promoter of the gene encoding the drug efflux pump ABCG2. Treating cultured CSCs or 4T1 tumor-bearing mice with the nonsteroidal anti-inflammatory drug aspirin restored SMAR1 expression and ABCG2 repression and enhanced tumor sensitivity to doxorubicin. Our findings reveal transcriptional mechanisms regulating SMAR1 that also regulate cancer stemness and chemoresistance and suggest that, by restoring SMAR1 expression, aspirin might enhance chemotherapeutic efficacy in patients with stem-like tumors.

Conventional chemotherapeutic regimens are unable to prevent metastasis of non-small cell lung carcinoma (NSCLC) thereby leaving cancer incurable. Cancer stem cells (CSCs) are considered to be the origin of this therapeutic limitation. In the present study we report that the migration potential of NSCLCs is linked to its CSC content. While cisplatin alone fails to inhibit the migration of CSC-enriched NSCLC spheroids, in a combination with non-steroidal anti inflammatory drug (NSAID) aspirin retards the same. A search for the underlying mechanism revealed that aspirin pre-treatment abrogates p300 binding both at TATA-box and initiator (INR) regions of mTOR promoter of CSCs, thereby impeding RNA polymerase II binding at those sites and repressing mTOR gene transcription. As a consequence of mTOR down-regulation, Akt is deactivated via dephosphorylation at Ser473 residue thereby activating Gsk3β that in turn causes destabilization of Snail and β-catenin, thus reverting epithelial to mesenchymal transition (EMT). However, alone aspirin fails to hinder migration since it does not inhibit the Integrin/Fak pathway, which is highly activated in NSCLC stem cells. On the other hand, in aspirin pre-treated CSCs, cisplatin stalls migration by hindering the integrin pathway. These results signify the efficacy of aspirin in sensitizing NSCLC stem cells towards the anti-migration effect of cisplatin. Cumulatively, our findings raise the possibility that aspirin might emerge as a promising drug in combinatorial therapy with the existing chemotherapeutic agents that fail to impede migration of NSCLC stem cells otherwise. This may consequently lead to the advancement of remedial outcome for the metastatic NSCLCs.

Cancer stem cells (CSCs) are a subset of cells within the tumor bulk, with a potential to undergo self-renewal, differentiation, proliferation and metastasis. Cumulative evidences suggest that mainly CSCs are responsible for tumor recurrences and resistance towards chemo- and radiotherapy. Unlike non-stem cancer cells, CSCs can undergo self-renewal and differentiation through asymmetric cell division (ACD) similar to normal stem cells. Asymmetric division results in two daughter cells: a structural and functional copy of the mother cell (CSC) and another one destined to undergo differentiation, non-cancer stem cell. Apart from the involvement of various polarity-determining factors, transcription factors and epigenetic regulators, micro-RNAs also play a crucial role in cell-fate decision making. Eventually, two daughter cells with distinct fates are produced with an unequal inheritance of differentiation-linked fate determinants, centrosomes and other stemness associated factors. Violation of the mechanisms leading to ACD will compromise the generation of both new CSCs and progenitor cells within the tumor. Thus, manipulation of asymmetric division of CSCs may introduce new strategies for dual targeting of CSCs and the tumor bulk. In this review, we discuss how CSCs play a critical role in almost all the events of tumorigenesis. Apart from that, we also delineate how these CSCs exploit the phenomenon of ACD during cancer relapses or tumor recurrences to regenerate the entire tumor. Therefore, in a nutshell this review highlights the therapeutic relevance of CSCs for successful elimination of cancer and suggests a novel improvisation in CSC-targeting by perturbing its ACD mechanisms.

Cancer development is initiated, sustained, and aggravated by a rare population of cells, termed cancer stem cells (CSCs). Although CSCs are considered as a promising source of cells to orchestrate the immune system to work in favour of tumor, the detailed mechanisms underlying their immunomodulatory effects remain elusive. Recent reports indicate the contribution of exosomes, secreted from various cells, as mediators of cell-to-cell communication especially within the tumor microenvironment. We aimed at exploring the role of CSC-derived exosomes (CDEs) in reprogramming the host immune system by generating functional T-regulatory (Treg) cells, and at delineating the underlying mechanisms. Our results showed that CDEs play a significant role in generating CD4 +CD25 +FoxP3 + Treg cells from naive T-cells. A search for the underlying mechanism revealed the presence of FoxP3 protein in CDEs which was found to be transferred to the naïve T-cells. Exosomes from FoxP3-ablated CSCs failed to augment immuno-suppressive Treg cell generation confirming the significant role of the transported protein. In order to understand the contribution of CDE-FoxP3 in maintaining a heritably stable population of Treg cell we checked for the binding of CDE-FoxP3 on conserved non-coding sequence 2 (CNS2) region of FoxP3 promoter in T-naïve cells and found CDE-FoxP3 is indeed recruited to the CNS2 region generating stable and functionally suppressive Treg cells. These results raise the possibility that CSCs provide the initial trigger for immunosuppressive Treg cell generation and thus, breaching the deadly-liaison between them might be a promising strategy in breast cancer therapy.

Background Well-known anti-malarial drug artemisinin exhibits potent anti-cancerous activities. In-vivo and in-vitro studies showed its anti-tumor and immunomodulatory properties signifying it as a potent drug candidate for study. The studies of mechanisms of cell movement are relevant which can be understood by knowing the involvement of genes in an effect of a drug. Although cytotoxicity and anti-proliferative activity of artemisinin is evident, the genes participating in its anti-migratory and reduced invasive effect are not well studied. The present study reports the alteration in the expression of 84 genes involved in cell motility upon artemisinin treatment in MCF-7 breast cancer cells using pathway focused gene expression PCR array. In addition, the effect of artemisinin on epigenetic modifier HDACs is studied. Methods We checked the functional stimulus of artemisinin on cell viability, migration, invasion and apoptosis in breast cancerous cell lines. Using qRT-PCR and western blot, we validated the altered expression of relevant genes associated with proliferation, migration, invasion, apoptosis and mammary gland development. Results Artemisinin inhibited cell proliferation of estrogen receptor negative breast cancer cells with fewer efficacies in comparison to estrogen receptor positive ones. At the same time, cell viability and proliferation of normal breast epithelial MCF10A cells was un-affected. Artemisinin strongly inhibited cancer cell migration and invasion. Along with orphan nuclear receptors (ERRα, ERRβ and ERRγ), artemisinin altered the ERα/ERβ/PR/Her expression status of MCF-7 cells. The expression of genes involved in the signaling pathways associated with proliferation, migration, invasion and apoptosis was significantly altered which cooperatively resulted into reduced growth promoting activities of breast cancer cells. Interestingly, artemisinin exhibited inhibitory effect on histone deacetylases (HDACs). Conclusions Upregulated expression of tumor suppressor genes along with reduced expression of oncogenes significantly associated with growth stimulating signaling pathways in response to artemisinin treatment suggests its efficacy as an effective drug in breast cancer treatment.

Background: The overexpression of oestrogen-related receptor- β (ERR β ) in breast cancer patients is correlated with improved prognosis and longer relapse-free survival, and the level of ERR β mRNA is inversely correlated with the S-phase fraction of cells from breast cancer patients. Methods: Chromatin immunoprecipitation (ChIP) cloning of ERR β transcriptional targets and gel supershift assays identified breast cancer amplified sequence 2 ( BCAS2 ) and Follistatin (FST) as two important downstream genes that help to regulate tumourigenesis. Confocal microscopy, co-immunoprecipitation (CoIP), western blotting and quantitative real-time PCR confirmed the involvement of ERR β in oestrogen signalling. Results: Overexpressed ERR β induced FST-mediated apoptosis in breast cancer cells, and E-cadherin expression was also enhanced through upregulation of FST. However, this anti-proliferative signalling function was challenged by ERR β -mediated BCAS2 upregulation, which inhibited FST transcription through the downregulation of β -catenin/TCF4 recruitment to the FST promoter. Interestingly, ERR β -mediated upregulation of BCAS2 downregulated the major G1-S transition marker cyclin D1, despite the predictable oncogenic properties of BCAS2. Interpretation: Our study provides the first evidence that ERR β , which is a coregulator of ER α also acts as a potential tumour-suppressor molecule in breast cancer. Our current report also provides novel insights into the entire cascade of ERR β signalling events, which may lead to BCAS2-mediated blockage of the G1/S transition and inhibition of the epithelial to mesenchymal transition through FST-mediated regulation of E-cadherin. Importantly, matrix metalloprotease 7, which is a classical mediator of metastasis and E-cadherin cleavage, was also restricted as a result of ERR β -mediated FST overexpression.

Abstract Crosstalk between thyroid and estrogen hormones actions have been documented with regard to a variety physiological functions. Both of these hormones regulate transcription of target genes by binding to their nuclear receptors that interact with specific responsive elements (estrogen and thyroid hormone response elements, i.e. ERE and TRE, respectively. Recently, we have demonstrated that 3, 3′5 Triiodo L Thyronine (T3) can induce apoptosis in ER+ve highly metastatic breast cancer MCF-7 cells through downregulating Senescence marker protein-30 (SMP30) gene. SMP30 has also been reported to be downregulated by 17β-Estradiol (E2) in prostrate gland and mammary epithelial cells. Interestingly, thyroid receptors (TRs) and estrogen receptor (ER) share a common response element, so we hypothesised a possible competition between both the receptors in SMP30 promoter under different hormone responses. To prove this hypothesis, gel retardation and luciferase assays were conducted by taking hSMP30 promoter reporter construct validated our findings of the putative ERE site. Competition Chromatin immunoprecipitation assay (ChIP) showed prominent TRβ binding irrespective of thyroid and estrogen hormone treatment. As a result, there was a significant repression found in whole promoter activity in presence of T3 and also in presence of both E2 and T3 treatment. Although the SMP30 promoter activity was same in response to E2 and T3 but the functional consequences after downregulation of SMP30 in human breast cancer cells were different. Overall our studies indicate that estrogen signalling/downstream target molecules of E2 weren't affected by estrogen mediated SMP30 downregulation however, SMP30 downregulation by thyroid hormone synergized the effect of T3 by further inducing apoptosis in human breast cancer cells. Thyroid hormone induced apoptosis in human breast cancer cells was through activated p53 molecule. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4961. doi:1538-7445.AM2012-4961

Abstract ERRβ regulates transcriptional activity of ERα as well as ERβ by modulating BCAS2 expression and leads to FST-mediated induction of nuclear fragmentation in ER-positive MCF-7 breast carcinoma cells Dharmendra K Bhargava*, Debomita Sengupta*, Sanjib Choudhary, Rosalima Peter, Dipti Ranjan Mishra, Sandip K Mishra. Corresponding Author *Equal Contribution Institute of Life Sciences (an institute under Department of Biotechnology, Govt. of INDIA), Nalco Square, Bhubaneswar, Odisha, INDIA Abstract: Estrogen related receptors (ERRs) are a group of nuclear receptors which are structurally and functionally related to Estrogen receptors, but do not bind to Estrogen. Diethylstilbestrol, which has been shown to act as a ligand of ERRs, can inhibit growth of ER-positive as well as Tamoxifen-resistant ER negative Breast Cancer cell lines and thus justifies the need of study of ERR target genes for therapeutic purpose. ERRβ has been shown to behave differently from ERRα and ERRγ in terms of regulating tumorigenesis. ERRβ expression level has been shown to be inversely co-related with S-phase fraction suggesting their role in inhibition of cellular proliferation. We have undertaken the current study to identify the common targets of Estrogen Receptors and ERR beta. Study of cross talk between ERRβ and ERα may unfold the mechanism of deregulation of ER alpha signaling leading to failure of cell cycle checkpoint induction or apoptosis in breast cancer cells. By chromatin immunoprecipitation cloning, we identified Breast Cancer Amplified Sequence 2 (BCAS2) and Follistatin (FST) to be two important target genes of ERRβ. Whereas BCAS2 is a coactivator of ER alpha as well as negative coregulator of p53, FST has been shown to enhance the ability of R30C breast carcinoma cells to undergo apoptosis and inhibit multi-organ metastasis of small cell lung carcinoma in natural-killer cell deprived SCID mice. Although, FST and BCAS2 are found to be targets of ERRβ, surprisingly FST expression is upregulated by 10 nM estrogen treatment, while BCAS2 is downregulated. This is indicative of involvement of ER alpha in the regulation of ERRβ target genes. The analysis of our western data revealed that FST activation is mediated by ERRβ. Further, our confocal analysis indicates that ERRβ is able to induce nuclear fragmentation in presence of estrogen in ER positive MCF-7 breast carcinoma cells. Additionally, our first time report on regulation of BCAS2 by ERRβ also provides direct evidence of modulation of ER α transcriptional activity by ERR beta. Our extensive study is clearly inclined towards the need of considering the prognostic importance of ERRβ in ER-positive breast tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-27. doi:1538-7445.AM2012-LB-27